1. Field of the Invention
This invention relates to the detection of microorganisms and the assessment of the phagocytosis of microorganisms. More particularly, it relates to the detection and the assessment of the phagocytosis of microorganisms in a blood sample, wherein the microorganisms are stained with a fluorescent dye and examined in an expanded buffy coat.
2. Description of the Prior Art
Blood is normally a sterile fluid. However, when microorganisms are present, the resulting infections may be life threatening situations. Successful treatment of blood infections requires early diagnosis, and proper treatment cannot be initiated until accurate identification of the pathogen has been accomplished. Identification is rendered very difficult in the early stages of the infection because the concentration of the pathogen in the blood is usually low.
A variety of systems has been proposed for the early detection of microorganisms in the blood. Two procedures are currently in use in hospital microbiology laboratories. One is a conventional blood culturing method wherein a growth medium is inocurlated with the patient's blood and an increase in turbidity, indicative of growth, is monitored over a period of time. This method is the one most utilized, but the length of time required for growth to result in turbidity is a severe disadvantage. In some instances, fastidious organisms may require up to 7 days for detection by turbidity.
The second hospital technique is an automated radioisotope detection method wherein the conversion of radioisotope .sup.14 C-labeled bacterial nutrients to .sup.14 C-labeled CO.sub.2 is monitored in the head space gas. This method has been shown to be able to detect 30% of the positive blood cultures in 12 hours and 70% within 24 hours, but is also dependent on bacterial growth for detection. Although detection is accomplished sooner than with the conventional culturing methods, the 12-24 hour period involved is still a serious drawback where rapid detection of the pathogen is important.
Culturing can sometimes be avoided if the microoganisms are concentrated or partially separated from some of the other components in the sample which might mask their presence and thereby hinder detection and subsequent identification. Standard methods for concentrating or separating microorganisms from such samples have relied on specific physical properties, such as size or density. However, use of such techniques is complicated by the fact that different species of microorganisms differ greatly in these physical properties. More recent methods allowing rapid identification include counterimmunoelectrophoresis, latex agglutination and radioimmunologic methods. Such methods, although successful in certain situations, have been much less successful in detection of circulating microorganisms, especially during the early stages of infection.
Other methods for the detection of microorganisms have employed various density gradients to separate the microorganisms from various blood components. Blood cell lysis/ filtration and lysis/centrifugation methods involve the addition of blood cell lysis agents to dispose of the eukaryotic cells and subsequent collection of the microorganisms by either passage through a bacterial retention membrane filter or by centrifugation. Exemplary of these methods is that disclosed in U.S. Pat. No. 4,131,512 to Dorn wherein a lysed blood sample is deposited on a high density liquid cushioning agent and subjected to centrifugation to cause collection of the bacteria at the interface between the cushioning agent and the sample. These methods, however, also require a final step of growing the bacteria on nutrient agar plates for detection by colony formation and thus are subject to the same limitation as direct culturing techniques.
Another method which aids in the isolation of microorganisms utilizes leukocytes. Leukocytes (white blood cells) include lymphocytes, monocytes, and granulocytes, and the term "leukocytes" as used herein referes to one or more of these subclasses.
One of the primary functions of leukocytes is ingestion and destruction of microorganisms invading the peripheral blood. The process of ingestion is known as phagocytosis and microorganisms which have been phagocytized are hereinafter referred to as intracellular bacteria. Microorganisms which have not been phagocytized are referred to as extracellular bacteria.
Phagocytosis is the natural defense mechanism against infection, and clinical microbiology laboratories have often sought to detect infections by visualizing and scoring leukocytes containing intracellular microorganisms. One such method which has achieved some limited success in serious blood infections, such as septicemia, is microscopic examination of smears made from the buffy coat. In this procedure, unclotted blood is centrifuged whereby the buffy coat forms from various blood components, including leukocytes, as a thin layer between the plasma and erythocyte layers. Microorganisms, if present, are also found in this layer and thus are concentrated and separated from some of the other blood components. Study of the buffy coat has heretofore required transfer from the centrifuge tube to a means of examination, as, for example, a microscope slide. Because of the narrowness of the buffy layer, such transfer has resulted in unavoidable errors.
U.S. Pat. No. 4,027,660 to Wardlow describes expansion of the buffy coat by the addition of a plastic float to a conventional microhematocrit tube wherein the plastic float is of the proper specific gravity so as to become positioned directly above the erythrocyte layer and directly below the plasma layer of a centrifuged blood sample. Thus it occupies the location of the buffy coat. Application of this device to obtain a complete blood cell count is described by Wardlaw et al. (J. Am. Med. Asso. 249, 617 (1983) and a commercial embodiment thereof is marketed (QBC Centrifugal Hematology System, Clay Adams, Division of Becton Dickinson & Co., Parsippany, N.J. Preliminary studies directed toward detection of blood borne parasites are also disclosed by Wardlaw et al.
Buffy coat smears may be stained with a variety of standard agents such as Gram, Wright, Jenner-Giemsa, Leishman, or May-Grunwald-Giemsa stains and visualized under the microscope. Recent studies (A. Kostiala et al., Am. J. Clin. Pathol. 72,437 (1981) have shown that, in rabbits, experimental bacteremias and fungemias induced by inoculations of a variety of pathogens could be detected regularly by microscopy or subculture of the Gram-stained buffy coat when cell populations reached 300-1000/ml. In contrast, Reik et al., J. Am. Med. Asso. 245, 357 (1981) report that bacteria in various samples, including blood, are not visible by Gram stain unless present in concentrations close to 1.times.10.sup.5 cells/ml. Also, in clinical studies, M. J. Coppen et al., (J. Clin. Pathol. 34, 1375 (1981) report that buffy coat smears have little practical value for identification of microorganisms because of the high incidence of false positivies and false negatives.
The use of fluorescence microscopy for a variety of purposes is increasing in both clinical and research laboratories, and has been used in a variety of inexpensive staining protocols. For example, ethidium bromide has been used extensively to stain both eukaryotic and prokaryotic cells. Acridine orange (AO) has been utilized to stain bacteria in various clinical samples, including blood (L. R. McCarthy and J. E. Senne, J. Clin. Microb., 11 281 (1980)). G. Kronvall and E. Myhre, (Acta Path. Microb. Scand, 85, 249 (1977)) describe AO staining of intracellular bacteria, but only buccal and traceal samples.
The analysis of phagocytosis has led to the elucidation of many syndromes with genetic or acquired defects in chemotaxis, motility, ingestion or microbial killing. Various methods to study phagocytic activity have been developed. These methods include treatment with agents which lyse extracellular particles while leaving intracellular particles intact, quantitation of the ingestion phase of phagocytosis by use of the electron microscope or radioactive agents, and differential staining of extracellular and intracellular microorganisms with fluorescent dyes. Exemplary of staining methods is that reported by Goldner (Laboratory Medicine, 14, 291 (1983)) wherein AO is used to stain all the microorganisms in a sample, which are counted. Subsequently, a second dye, crystal violet, is used to quench the fluorescence in the external environment, leaving the intracellular microorganisms as the only fluorescing species. A count of extracellular microorganims is obtained by difference.
The above methods require expensive equipment, use of dangerous radioactive agents, or require excessive operator handling. There is a definite need for a better method for the assessment of phagocytic activity in a biological fluid sample.